Tissue regenerative composition, method of making, and method of use thereof

ABSTRACT

A matrix, including epithelial basement membrane, for inducing repair of mammalian tissue defects and in vitro cell propagation derived from epithelial tissues of a warm-blooded vertebrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims priority to U.S.provisional patent application serial No. 60/171,733, filed Dec. 22,1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was supported under National Institutes of HealthGrants, 1-R43HL85 761-01, 1-R43-DC-04387-01, and 1-R43-DC-4387-02.

TECHNICAL FIELD

[0003] This invention relates to devitalized acellular tissueregeneration compositions, methods of making, and methods of use.

BACKGROUND OF THE INVENTION

[0004] Submucosal tissues of warm-blooded vertebrates are useful intissue grafting materials. For example, submucosal tissue graftcompositions derived from small intestine have been described in U.S.Pat. No. 4,902,508 (hereinafter the '508 patent) and U.S. Pat. No.4,956,178 (hereinafter the '178 patent), and submucosal tissue graftcompositions derived from urinary bladder have been described in U.S.Pat. No. 5,554,389 (hereinafter the '389 patent). All of thesecompositions consist essentially of the same tissue layers and areprepared by the same method, the difference being that the startingmaterial is small intestine on the one hand and urinary bladder on theother. The procedure detailed in the '508 patent, incorporated byreference in the '389 patent and the procedure detailed in the '178patent, includes mechanical abrading steps to remove the inner layers ofthe tissue, including at least the luminal portion of the tunica mucosaof the intestine or bladder, i.e., the lamina epithelialis mucosa(epithelium) and lamina propria, as detailed in the '178 patent.Abrasion, peeling, or scraping the mucosa delaminates the epithelialcells and their associated basement membrane, and most of the laminapropria, at least to the level of a layer of organized dense connectivetissue, the stratum compactum. Thus, the tissue graft materialpreviously recognized as soft tissue replacement material is devoid ofepithelial basement membrane and consists of the submucosa and stratumcompactum.

[0005] The epithelial basement membrane is a thin sheet of extracellularmaterial contiguous with the basilar aspect of epithelial cells. Sheetsof aggregated epithelial cells of similar type form an epithelium.Epithelial cells and their associated epithelial basement membrane arepositioned on the luminal portion of the tunica mucosa and constitutethe internal surface of tubular and hollow organs and tissues of thebody. Epithelial cells and their associated epithelial basement membraneare also positioned on the external surface of the body, i.e., skin.Examples of a typical epithelium having a basement membrane include, butare not limited to the following: the epithelium of the skin, intestine,urinary bladder, esophagus, stomach, cornea, and liver.

[0006] Epithelial cells are positioned on the luminal or superficialside of the epithelial basement membrane, opposite to connectivetissues. Connective tissues, the submucosa, for example, are positionedon the abluminal or deep side of the basement membrane. Examples ofconnective tissues that are positioned on the abluminal side of theepithelial basement membrane are the submucosa of the intestine andurinary bladder, and the dermis and subcutaneous tissues of the skin.

SUMMARY OF THE INVENTION

[0007] The present invention provides devitalized tissue regenerativecompositions comprising an epithelial basement membrane as part of amatrix or scaffold for tissue repair or regeneration. The inclusion ofthe epithelial basement membrane in devitalized mammalian tissueregenerative compositions results in improved in vivo endogenous cellpropagation and tissue restoration as compared to submucosal matricesdescribed above which do not include an epithelial basement membrane.For the purposes of this invention, devitalized means acellular orsubstantially acellular. For the purposes of this invention, epithelialbasement membrane means at least a portion of the intact epithelialbasement membrane.

[0008] According to the invention, a preferred devitalized matrix formammalian tissue repair or regeneration comprises at least a portion ofa mammalian epithelial basement membrane, preferably the entireepithelial basement membrane, and the tunica propria that is immediatelysubjacent to the basement membrane. Devitalized matrices of theinvention restore or replace diseased, defective, or missing tissue whenplaced in contact with host tissue. In a preferred embodiment, theinvention comprises a devitalized matrix that is custom-shaped toconform to the diseased or defective tissue. In a particular embodiment,the matrix comprises a sheet of matrix derived from the urinary bladder,the intestine, or any other mammalian epithelial tissue. In anotherembodiment, the matrix is injectable by means of being transformed intoa fine particulate, emulsion, gel or extract. A matrix of the inventionmay act as a carrier for a pharmaceutical agent. A preferred applicationof the matrix of the invention is the repair or restoration of cardiactissue. In particular, a matrix or composition of the invention isuseful to restore or replace at least a portion of a cardiac valve, theinteratrial septum, the interventricular septum, or the myocardium. Forthe purposes of this invention, matrix and composition areinterchangeable terms.

[0009] In one embodiment, the invention features a devitalizedcomposition comprising epithelial basement membrane and tunica propriaimmediately subjacent to the basement membrane. The epithelial basementmembrane and tunica propria immediately subjacent to the basementmembrane are delaminated from cells of a mammalian epithelium andabluminal portions of the tunica propria. Mammalian epithelial tissueused in this aspect of the invention is preferably derived from urinarybladder, intestine, or any other mammalian epithelial tissue. Furtherembodiments feature a composition shaped to conform to a diseased ordefective cardiac valve such as at least a portion of a pulmonic valve,aortic valve, right or left atrioventricular valve, or the myocardium.

[0010] In still another embodiment, the invention features a compositioncomprising epithelial basement membrane, tunica propria, and submucosa.The epithelial basement membrane and tunica propria are delaminated fromthe cells of an epithelium and from the tunica muscularis of a mammalianepithelial tissue.

[0011] In yet another embodiment, the invention features a compositioncomprising epithelial basement membrane, tunica propria, and smoothmuscle cells of the tunica muscularis, all delaminated from epithelialcells of a mammalian epithelium.

[0012] The composition, according to the invention, is not limited tomerely the embodiments enclosed. Rather, the composition, according tothe invention, comprises one or more layers of an epithelial tissue incombination with at least a portion, preferably the entire, intactepithelial basement membrane.

[0013] In another aspect, the invention provides methods for inducingrestoration or repair of diseased or defective cardiac tissue. Apreferred method of the invention comprises the step of contacting ahost tissue with a devitalized matrix derived from a mammal. Thedevitalized matrix comprises at least a portion of an epithelialbasement membrane and tunica propria immediately subjacent to thebasement membrane. In preferred embodiments, methods of the inventioncomprise inducing endogenous epithelial repair using tissue regenerativecompositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings are not to scale and emphasis instead is generallybeing placed upon illustrating the principles of the invention.

[0015]FIG. 1A is a cross-sectional view of the wall of the intestine.

[0016]FIG. 1B is a cross-sectional view of the wall of the urinarybladder.

DETAILED DESCRIPTION OF THE INVENTION

[0017] A devitalized tissue regenerative composition in accordance withthe present invention comprises epithelial basement membrane or at leasta portion of the epithelial basement membrane and at least the subjacentportion of the tunica propria harvested from a mammalian epithelialtissue. Preferred epithelial tissues for use in the invention include,but are not limited to, urinary bladder and other tissues of theuro-genital tract, small intestine, esophagus and other tissues of thegastrointestinal tract, skin, liver, and arteries such as the aorta andother tissues of the cardiovascular system. In a preferred embodiment,the invention provides a tissue graft composition comprising at least aportion of the epithelial basement membrane and subjacent tunicapropria, separated from the luminal epithelial cells, the abluminaladventitial, serosal, and smooth muscle layers and the submucosal tissuelayers Tissue separation or delamination techniques, according to theinvention, provide a layer of devitalized extracellular matrix materialincluding epithelial basement membrane or at least a portion of theepithelial basement membrane essentially free of cells. Any remainingcellular elements are then removed by further processing steps such asrinsing in hypotonic saline, peracetic acid or sterile water.

[0018] Accordingly, referring to FIGS. 1A and 1B, a preferred embodimentof the invention comprises epithelial basement membrane B and thebiotropic connective tissue known as the tunica propria C that isimmediately subjacent to and positioned on the abluminal side of theepithelial basement membrane B of the intestine illustrated in FIG. 1A,or the urinary bladder illustrated in FIG. 1B or any other epithelialtissue. This embodiment of the invention features the epithelialbasement membrane B and portions of the tunica propria C adjacent to theepithelial basement membrane B. The epithelial basement membrane B andtunica propria C are delaminated from the epithelial cells A, thesubmucosa D, the tunica muscularis E, and the serosa F. Thus, in thisembodiment of the invention, the portions of the tunica mucosa Hadjacent the lumen L, i.e., the luminal portions of the tunica mucosa,form a preferred tissue matrix composition.

[0019] In another preferred embodiment, again referring to FIGS. 1A and1B, a composition of the invention comprises epithelial basementmembrane B, tunica propria C, and the tunica submucosa D. The epithelialbasement membrane B, tunica propria C, and the tunica submucosa D aredelaminated from the epithelial cells A, tunica muscularis E, and tunicaserosa F. In this embodiment, the portions of the tunica mucosa H thatinclude the epithelial basement membrane, and the tunica submucosa, forma preferred tissue matrix composition.

[0020] In yet another composition, a preferred embodiment of theinvention comprises the epithelial basement membrane B, the tunicapropria C that lie adjacent the epithelial basement membrane B, thetunica submucosa D and at least a portion of the tunica muscularis E.

[0021] Sources of Epithelial Tissue

[0022] Material for tissue regeneration compositions of the invention istypically prepared from tissue harvested from animals raised for meatproduction, including but not limited to, pigs, cattle and sheep. Otherwarm-blooded vertebrates are also useful as a source of tissue, but thegreater availability of such tissues from animals used for meatproduction makes such tissue preferable. Thus, there are inexpensivecommercial sources of tissue for use in preparation of the tissuecompositions in accordance with the present invention. There may bespecially bred or genetically engineered strains of certain species thatare used as a tissue source. For example, pigs that are geneticallyengineered to be free of the galacatosyl, alpha 1,3 galactose (GALepitope) may be used as the source of tissues for production of thecomposition. Alternatively, herds of pigs that are raised to be free ofspecific pathogens may be used as the source of tissues. Mammaliantissue used as the source of tissue for production of the composition ofthe invention may be harvested from an animal of any age group,including embryonic tissues, market weight, gender or stage of sexualmaturity.

[0023] Tissue Sources of Epithelial Basement Membrane

[0024] Urinary Bladder

[0025] A preferred source of epithelial basement membrane is the urinarybladder illustrated in FIG. 1B of a warm-blooded vertebrate such as apig. Superior biologic tissue remodeling properties are derived fromepithelial basement membrane components that support and promote cellgrowth without invasion, and the subjacent tunica propria matrixmaterial that allows and promotes endogenous cellular adhesion,invasion, growth and differentiation. The matrix, referred tohereinafter as urinary bladder matrix (UBM), includes the basementmembrane B of the urinary bladder epithelium and the subjacent tunicapropria C. In this embodiment, epithelial basement membrane B and thesubjacent tunica propria C are delaminated from the epithelial cells Aand the extracellular matrix of the tunica submucosa D, the tunicamuscularis E, and the tunica serosa F. UBM is harvested from anywarm-blooded vertebrate but is most preferably harvested from pigs. UBMis used as a bioscaffold for the repair or restoration of body tissuesand organs such as musculoskeletal and cardiovascular structures,dermatologic and gastrointestinal tissues, urogenital and reproductivetissues, neurologic tissues, liver, kidney, and head and neck tissues.

[0026] A preferred UBM tissue regenerative composition comprisesepithelial basement membrane, preferably urinary bladder basementmembrane, and the biotropic molecular structure that lies immediatelysubjacent the epithelial basement membrane from the urinary bladdertissue of warm blooded vertebrates. In this embodiment, epithelialbasement membrane is delaminated from the luminal epithelial cells,abluminal adventitial, serosal, and smooth muscle tissues, andsubmucosal tissues. Tissue graft compositions of the invention haveremarkably superior tissue growth characteristics as compared topreviously described submucosal tissue graft compositions implanted orinjected into a vertebrate host to cause the repair or replacement ofdamaged, missing, or defective tissues or organs.

[0027] Methods of the present invention avoid complete loss of theepithelial basement membrane and result in a tissue regenerativecomposition that includes at least a portion of the epithelial basementmembrane. In a preferred embodiment, the epithelial basement membrane asdetermined by conventional histochemical or immunohistochemicaltechniques and light, or electron microscopy, is largely intact. Theresulting devitalized material obtained by methods of the presentinvention is in contrast to methods for making tissue graft compositionsderived from small intestine and urinary bladder as described in the'508 and '389 patents which result in a graft material includingsubmucosa exclusive of the epithelial basement membrane. Steps inpreparation of UBM from urinary bladder tissue differ from previouslydescribed steps for preparation of submucosal tissue graft compositiondescribed in the '508 patent and the '389 patent. In the methods for thepreparation of the submucosal tissue graft composition described in the'508 and '389 patents, the mucosa is mechanically removed by abrasion.

[0028] According to the present invention, UBM is prepared by removingthe urinary bladder tissue from a warm-blooded vertebrate, for example,a pig, and delaminating the tissue by first soaking the tissue in adeepithelializing solution, for example, hypertonic saline, mostpreferably 1.0 N saline, for periods of time ranging from 10 minutes to4 hours. Exposure to hypertonic saline solution effectively removes theepithelial cells from the underlying basement membrane. The tissueremaining after the initial delamination procedure includes epithelialbasement membrane and the tissue layers abluminal to the epithelialbasement membrane. This tissue is next subjected to further treatment toremove the majority of abluminal tissues but not the epithelial basementmembrane. The outer serosal, adventitial, smooth muscle tissues,submucosa and abluminal portion of the tunica propria are removed fromthe remaining deepithelialized tissue by mechanical abrasion or by acombination of enzymatic treatment, hydration, and abrasion. Mechanicalremoval of these tissues is accomplished by removal of mesenterictissues with, for example, Adson-Brown forceps and Metzenbaum scissorsand wiping away the tunica muscularis and abluminal tunica propria usinga longitudinal wiping motion with a scalpel handle or other rigid objectwrapped in moistened gauze. After these tissues are removed, theresulting tissue scaffold consists of epithelial basement membrane andsubjacent tunica propria. This tissue differs from previously knowntissue compositions derived from animal epithelial tissues by theinclusion of a largely intact epithelial basement membrane in thepresent invention. The tissues may be further processed by rinsing inhypertonic saline, peracetic acid or sterile water. Other methods forremoving tissue layers, a microtome, for example, may also be used toobtain the tissue composition of the invention.

[0029] The method for preparation of tissue regenerative compositionsaccording to the invention is not limited to the use of urinary bladdertissue as a starting material. The method according to the invention isalso applicable to other starting tissues, for example, skin, esophagus,stomach, and intestinal tissues.

[0030] After preparing UBM according to the method of the invention, theresulting tissue scaffold consists of an approximately 10-120 micrometerthick material that consists primarily (i.e., greater than 90%) ofextracellular matrix (ECM) including the epithelial basement membrane.This material may or may not retain some of the cellular elements thatcomprised the original tissue such as capillary endothelial cells orfibrocytes. These cellular elements are removed by subsequent exposureto peracetic acid as part of the disinfection of the biomaterial. Thematerial differs in its histologic appearance and its architecture fromthe submucosal tissue graft compositions because of the smoothepithelial basement membrane that demarks the luminal surface and thedense, partially organized collagenous ECM that demarks the abluminalsurface. The ECM material stains pink with H&E stain and blue withMasson's trichrome stain.

[0031] Skin Esophagus

[0032] Similarly, steps used in preparation of tissue regenerativecompositions from other epithelial organs having tissue layers similarto urinary bladder, such as skin, or esophagus, parallel the stepsdescribed above for preparing UBM. Like the urinary bladder matrix, thematerial remaining after removal of the epithelial cells, tunica serosa,tunica muscularis and abluminal tunica propria, includes at least aportion of the epithelial basement membrane, and the adjacent tunicapropria.

[0033] Small Intestine

[0034] A tissue regenerative composition of the invention is alsoderived from epithelial tissues of the gastrointestinal tract, such asthe small intestine. Steps in preparation of a tissue regenerativecomposition that includes at least a portion of the epithelial basementmembrane of the small intestine and subjacent tunica propria, termedSIM, are similar to the steps described above for the formation of UBM.1.0N saline may be used to remove the intestinal epithelial cells fromthe epithelial basement membrane. An alternate method for removingepithelial cells is to soak the epithelial tissue in a detergent such asa non-ionic detergent, for example, Triton X-100, at concentrations from0.025 to 1%, for 5 minutes to several hours.

[0035] In one embodiment, the delaminated tissue regenerativecomposition derived from an epithelial tissue is stored either in afrozen hydrated state or is air dried at room temperature, then stored.Alternatively, the tissue regenerative composition is lyophilized andstored in a dehydrated state at either room temperature or frozen. Inyet another embodiment, the tissue regenerative composition can beminced and fluidized by digesting the material in proteases, for examplepepsin or trypsin, for periods of time sufficient to solubilize thetissue and form a substantially homogeneous solution. The viscosity ofthe solubilized material can be varied by adjusting the pH to create agel, gelsol, or completely liquid state. The preparation of fluidizedintestinal submucosa, for example, is described in U.S. Pat. No.5,275,826, expressly incorporated herein by reference.

[0036] In still another embodiment, the present invention contemplatesthe use of powder forms of the tissue regenerative composition. In oneembodiment, a powder form of tissue regenerative composition is createdby mincing or crushing the delaminated material to produce particlesranging in size from 0.005 mm² to 2.0 mm². The material, delaminatedfrom unwanted tissue layers, is frozen for example, in liquid nitrogen,to perform the crushing procedure. Alternatively, the material isdehydrated to perform the crushing procedure. The crushed form of thematerial is then lyophilized to form a substantially anhydrousparticulate tissue regenerative composition.

[0037] Tissue compositions of the present invention are suitable formany surgical and nonsurgical applications for the purpose of inducingreconstructive wound healing and tissue restoration. For example, theyare used to replace damaged, diseased, or missing heart valves,arteries, veins, urinary bladder, liver, portions of thegastrointestinal tract, or they can be used as templates for repair orreplacement of head and neck structures. The material, in any of anumber of its solid or fluidized forms, can be used as a scaffold fordermal or epidermal repair, injected into various body sphincters suchas urinary sphincter or esophageal or gastric sphincters, folded into atube or partial tube as a conduit for the restoration of nervous tissueor extruded or molded into any shape suitable for its application as atissue regenerative composition. The tissue regenerative composition ofthe invention can be sutured into place in its solid sheet form, placedin wounds or body locations in a gel form, or injected in its liquid orparticulate form. Tissue compositions of the present invention inducegrowth of endogenous tissues including epithelial and connective tissueswhen target tissues in vivo are placed in contact with mammalianderived, devitalized tissue compositions comprising at least a portionof an epithelial basement membrane.

[0038] Urinary Bladder Matrix (UBM)

[0039] UBM compositions comprise at least type I and type IV collagen,glycosaminoglycans, including hyaluronic acid, chondroitin sulfate A andB, heparin and heparin sulfate. In addition, one or more of basicfibroblast growth factor, vascular endothelial cell growth factor andTGF-beta are present in UBM.

[0040] The physical properties of UBM have been partially characterized.UBM has a uniaxial strength of approximately 0.1-2.0 pound per 1.0 cmwide strip (measured with a material testing system machine via AmericanStandards for Testing Materials pulling at 1 inch/minute). The sutureretention strength of the material is approximately 1.0-4.0 Newtons (N)per sheet layer, specifically, 4-18 N for a 4 layer matrix and 30 N-120N for a 30 layer matrix. The ball burst test failure force isapproximately 4-10 pounds for each layer, specifically, 32-80 N for 8layers, 16-40 N for 4 layers, and 36-120 N for 12 layers.

[0041] The porosity index is defined as the amount of water that flowsthrough a material per cm²/minute at 120 mmHg pressure. Water porositydiffers from one side of UBM to the other depending on the direction offlow. Water flows from the epithelial basement membrane to the abluminalside at approximately 20% the rate of water flow from the abluminal sideto the epithelial basement membrane side of the matrix. UBM also hasviscoelastic properties.

[0042] UBM can be sterilized by any of a number of standard methodswithout loss of its ability to induce endogenous tissue growth. Forexample, the material, after rinsing in saline and peracetic acid at0.05% to 1.0%, can be sterilized by ethylene oxide treatment, gammairradiation treatment (0.5 to 2.5 mRad), gas plasma sterilization, ore-beam treatment. The material can also be sterilized by treatment withglutaraldehyde that causes cross linking of the protein material, butthis treatment substantially alters the material such that it is slowlyresorbed or not resorbed at all and incites a different type of hostremodeling which more closely resembles scar tissue formation orencapsulation rather than constructive remodeling. Cross-linking of theprotein material can also be induced with carbodiimide or dehydrothermalor photooxidation methods.

[0043] The following examples will serve to better demonstrate thesuccessful practice of the present invention.

Exemplification

[0044] As exemplification of the utility of methods and compositions ofthe invention, UBM is applied to heart valve defects. As will beappreciated by those of ordinary skill in the art, methods andcompositions disclosed herein are applicable to other tissueregenerative compositions derived from sources of epithelial tissueother than the urinary bladder, from mammalian sources other than pigs,and to tissue defects other than heart valve. Moreover, tissueregenerative composition of the invention can be applied in a form otherthan a sheet or multilayer sheet of material, for example, UBM may beapplied as an extract, in gel form, powdered form, tubular form, sheetform, or as strips, cords or struts or mixed with other pharmaceuticalagents, for example, growth factors and gene products. UBM may beextruded or molded in or on a form to fit a particular application inthe body. The preparation of fluidized forms of tissue is described inU.S. Pat. No. 5,275,826, the disclosure of which is incorporated hereinby reference, and the preparation of solid sheets and strips of tissueis described in U.S. Pat. No. 5,711,969, the disclosure of which isincorporated herein by reference.

[0045] Application 1: Cardiac Tissue Repair

[0046] One embodiment, according to the invention, is a tissueregenerative composition for repair or replacement of cardiac tissues.Cardiac tissues include, but are not limited to, diseased, damaged, ormissing heart tissue including myocardium, epicardium, endocardium,pericardium, interatrial and interventricular septum and all heartvalves and associated valve leaflets including pulmonic valve, aorticvalve, right atrioventricular valve and left atrioventricular valve andportions of adjacent vessels of the heart including pulmonary artery,pulmonary vein, aorta, inferior vena cava, and superior vena cava.

[0047] In this embodiment of the invention disclosed herein, UBM wasprepared from porcine urinary bladder as described above, and used asautogenic and xenogenic anterior heart valve replacement leaflet of thepulmonic valve in five pigs and three dogs.

[0048] UBM, configured as a single sheet of material or as doublethickness material, was cut with scissors or a scalpel at the time ofsurgery to fit the pulmonic valve anterior leaflet. UBM was sutureddirectly to the annulus at the base of the valve. In the single sheetembodiment, the epithelial basement membrane side of UBM was positionedon the right ventricular luminal side of the replacement valve leafletand sutured directly to the annulus of the pulmonic valve. In a doublethickness embodiment of UBM, UBM was folded so that the epithelialbasement membrane was positioned on both surfaces, i.e., ventricular andarterial surfaces, of the replacement pulmonic valve leaflet, andsutured directly to the annulus of the pulmonic valve.

[0049] The pulmonic valves of experimental dogs and pigs were examined 6and 12 weeks after valve replacement. One dog was examined at 5 monthsafter valve leaflet replacement. Standard tissue fixation andhistopathological techniques were used to examine the harvested valveleaflets.

[0050] At six weeks post valve leaflet replacement, epithelialization ofthe replacement valve leaflet was present over the entire valve leafletsurface. Cells migrating over the valve leaflet surface stained positiveby immunofluorescent staining for von Willebrand factor indicating thatthese cells were of endothelial origin. In some valve leaflets some ofthe endothelial cells had features of early progenitor cells.Neovascularization, endothelial cell infiltration, and deposition ofextracellular matrix were observed originating from the host tissue atthe annulus of the pulmonic valve and extending into the replacementvalve leaflet.

[0051] At twelve weeks and at five months post valve leafletreplacement, none of the original UBM tissue composition wasrecognizable and restoration of the valve leaflet was complete.Unexpected findings at all time points examined included lack ofendothelial invasion into the replacement valve leaflet, lack ofthrombosis, and lack of calcification or cell-mediated rejection of thereplacement valve leaflet. Moreover, the shape of the replacement valveleaflet was unchanged from the shape of the original valve leaflet, atall time points examined.

[0052] Ultrasound studies of the pulmonic valve in pigs at 8, 12, 16 and20 weeks after valve replacement demonstrated a competent valve.

[0053] In another embodiment of this aspect of the invention, fluidized,powderized, or pulverized forms of UBM are applied to or injected intoor adjacent to diseased or defective cardiac tissue to promoteendogenous tissue repair. For example, fluidized UBM is injected into oradjacent to a congenital interventricular septal defect, congenitalinteratrial septal defect, or into the lumen of a patent ductusarteriosus to promote endogenous growth of tissue in these areas.

[0054] Application of UBM to cardiac tissues is accomplished by theapplication of several different surgical approaches. For example, aminimal invasive procedure is used to approach the cardiac surgical sitewith the aid of a laproscope. Alternatively, a thoracotomy is performed.UBM is brought to the surgical site in any of its prepared forms such asa sheet, loop, strip or as an injectable, powered, or pulverized form.Sheets or strips of UBM are custom-fit for the particular cardiacapplication before or during the surgical procedure. Sheets or strips ofUBM are secured adjacent to or in the defective or diseased cardiactissue with sutures, staples, tissue glue, or any other means known toone skilled in the art.

[0055] Application 2: Matrix for In Vitro Cell Proliferation

[0056] Human microvascular endothelial cells (HMVEC) form endothelium, asingle layer of cells organized on a basement membrane in vivo in amanner that mimics epithelium. Studies were conducted in vitro usingisolated HMVEC plated on (i) the epithelial basement membrane side of asheet of UBM, (ii) the abluminal surface of UBM, (iii) small intestinesubmucosa tissue graft composition (SIS) prepared according to themethods disclosed in the '508 and '178 patents, and (iv) urinary bladdersubmucosa tissue composition (UBS) prepared according the methoddisclosed in the '389 patent.

[0057] HMVEC grew into the matrix and did not form a confluent celllayer following three days' growth when plated on the surface of SIS andUBS regardless of whether HMVEC were plated on the luminal or abluminalsurface of SIS or UBS.

[0058] HMVEC plated on the abluminal surface of UBM grew into thematrix, proliferated and differentiated into mature endothelial cells.Like HMVEC plated on the abluminal surface of SIS and UBS, a confluentlayer of HMVEC was not formed on the abluminal surface of UBM followingthree days' growth.

[0059] In contrast to other previously known tissue regenerativecompositions such as SIS and UBS in these studies, HMVEC plated on theepithelial basement membrane side (luminal) of a sheet of UBM attachedto UBM, proliferated, differentiated and formed a confluent monolayerfollowing three days' growth.

[0060] Application 3

[0061] It is contemplated that the tissue graft composition of thepresent invention can be used to induce repair or replacement of tissuein vivo, including connective tissues, such as ligaments, tendons,cartilage, bone, joints, and muscle, epithelial tissues, such as urinarybladder, and other tissues of the urogenital tract, stomach, esophagus,and other tissues of the gastrointestinal tract, liver, nervous tissue,tissues of the head and neck, skin, and other tissues using the sameprocedures described in U.S. Pat. Nos. 4,902,508; 4,956,178; 5,281,422;5,352,463; 5,554,389; 5,275,826; 4,902,508; 5,372,821; 5,445,833;5,516,533; 5,573,784; 5,641,518; 5,695,998; 5,711,969; 5,755,791;5,762,966; and 5,885,619, the disclosures of which are incorporatedherein by reference. The tissue graft composition of the invention canalso be used with synthetic or non-synthetic polymers for restoration oftissues.

We claim:
 1. A devitalized matrix for inducing repair of tissue defectsin a mammal, comprising: an isolated devitalized mammalian epithelialbasement membrane and tunica propria immediately subjacent to saidbasement membrane.
 2. The matrix of claim 1 wherein said basementmembrane is derived from urinary bladder.
 3. The matrix of claim 1wherein said basement membrane is derived from small intestine.
 4. Thematrix of claim 1 wherein said matrix is sutured at said tissue defect.5. The matrix of claim 1 wherein said matrix is injected into saidtissue defect.
 6. The matrix of claim 1 wherein said matrix is appliedto a tissue defect fixation device.
 7. The matrix of claim 1 whereinsaid matrix is mixed with a pharmaceutical agent.
 8. A method forinducing repair of a tissue defect in a mammal, said method comprising:providing to a defect site, a devitalized matrix comprising a mammalianepithelial basement membrane and tunica propria immediately subjacent tosaid basement membrane.
 9. The method of claim 8 wherein said repaircomprises induction of connective tissue repair and epithelial tissuerepair at said tissue defect site.
 10. A devitalized composition,comprising: mammalian epithelial basement membrane and tunica propriaimmediately subjacent to said epithelial basement membrane, wherein saidbasement membrane and tunica propria are delaminated from cells of anepithelium and abluminal portions of a tunica propria of a mammalianepithelial tissue.
 11. A devitalized composition, comprising: mammalianepithelial basement membrane, tunica propria, and smooth muscle cells ofthe tunica muscularis, wherein said basement membrane, tunica propria,and smooth muscle are delaminated from epithelial cells of a mammalianepithelium.
 12. A devitalized composition, comprising: mammalianepithelial basement membrane and submucosa, wherein said basementmembrane and submucosa are delaminated from the cells of an epitheliumand tunica muscularis of a mammalian epithelial tissue.
 13. A method formanufacture of a devitalized tissue graft composition, comprising:soaking a mammalian epithelial tissue in a deepthelializing solution toform a deepithelialized tissue having an epithelial basement membrane;and, abrading said deepithelialized tissue on an abluminal surface ofsaid tissue to form a delaminated tissue, wherein the delaminated tissueremaining after abrading said tissue, comprises at least a portion ofthe epithelial basement membrane and said delaminated tissue comprisingsaid basement membrane induces endogenous tissue restoration.
 14. Themethod of claim 13 wherein said deepithelializing solution comprises 1.0N saline.
 15. The method of claim 13 wherein said abluminal surfacecomprises a tissue surface deeper than said epithelial basementmembrane.
 16. The method of claim 13 wherein said epithelial tissuecomprises urinary bladder.
 17. The method of claim 13 wherein saidepithelial tissue comprises small intestine.